annotate

CIRCexplorer2 annotate integrates CIRCexplorer into CIRCexplorer2. It annotates back-splicing junction reads with user provided gene annotations.

Usage and option summary

Usage:

CIRCexplorer2 annotate [options] -r REF -g GENOME -b JUNC [-o OUT]

Options:

-h --help                      Show help message.
--version                      Show version.
-r REF --ref=REF               Gene annotation.
-g GENOME --genome=GENOME      Genome FASTA file.
-b JUNC --bed=JUNC             Input file.
-o OUT --output=OUT            Output file. [default: circularRNA_known.txt]
--no-fix                       No-fix mode (useful for species with poor gene annotations).
--low-confidence               Extract low confidence circRNAs.

Notes about options

1. It would randomly report one circular RNA isoform for each back-splicing junction based on existed gene annotations.
2. If you set --no-fix options, realignment step of fusion junction reads will be skipped. It is useful for species with poor gene annotations, but the accuracy of circular RNA prediction would decrease.
3. CIRCexplorer2 annotate extracts fusion junction reads exactly matching the boundaries of exons of the same isoform by default. If you set the --low-confidence, it will also extract fusion junction reads matching the boundaries of exons of the different isofoms of the same gene, and output them in low_conf_circularRNA_known.txt.

Input

CIRCexplorer2 annotate needs a gene annotation file, a reference genome sequence file and a back_spliced_junction.bed created by CIRCexplorer2 parse or CIRCexplorer2 align.

back_spliced_junction.bed

Format of gene annotation file:

The file is in the format of Gene Predictions and RefSeq Genes with Gene Names below. (See example file)

Field	Description
geneName	Name of gene
isoformName	Name of isoform
chrom	Reference sequence
strand	+ or - for strand
txStart	Transcription start position
txEnd	Transcription end position
cdsStart	Coding region start
cdsEnd	Coding region end
exonCount	Number of exons
exonStarts	Exon start positions
exonEnds	Exon end positions

Output

CIRCexplorer2 annotate will create a circularRNA_known.txt file by default. The circularRNA_known.txt contains the final circular RNA annotation information.

circularRNA_known.txt
low_conf_circularRNA_known.txt

• circularRNA_known.txt: Circular RNA annotation file.

Format of circularRNA_known.txt:

Field	Description
chrom	Chromosome
start	Start of circular RNA
end	End of circular RNA
name	Circular RNA/Junction reads
score	Flag of fusion junction realignment
strand	+ or - for strand
thickStart	No meaning
thickEnd	No meaning
itemRgb	0,0,0
exonCount	Number of exons
exonSizes	Exon sizes
exonOffsets	Exon offsets
readNumber	Number of junction reads
circType	Type of circular RNA
geneName	Name of gene
isoformName	Name of isoform
index	Index of exon or intron
flankIntron	Left intron/Right intron

Format of low_conf_circularRNA_known.txt:

Field	Description
chrom	Chromosome
start	Start of circular RNA
end	End of circular RNA
name	Circular RNA/Junction reads
score	Flag of fusion junction realignment
strand	+ or - for strand
leftInfo	Gene:Isoform:Index of left exon
rightInfo	Gene:Isoform:Index of right exon